BACKGROUND OF THE INVENTION
Field of the Invention
Nucleic acid hybridization has been employed for
investigating the identity and establishing the presence of
nucleic acids. Hybridization is based on complementary base
pairing. When complementary single stranded nucleic acids are
incubated together, the complementary base sequences pair to
form double stranded hybrid molecules. The ability of single
stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid
(RNA) to form a hydrogen bonded structure with a complementary
nucleic acid sequence has been employed as an analytical tool
in molecular biology research. The availability of
radioactive nucleoside triphosphates of high specific activity
and the 32P labelling of DNA with T4 polynucleotide kinase has
made it possible to identify, isolate, and characterize
various nucleic acid sequences of biological interest.
Nucleic acid hybridization has great potential in diagnosing
disease states associated with unique nucleic acid sequences.
These unique nucleic acid sequences may result from genetic or
environmental change in DNA by insertions, deletions, point
mutations, or by acquiring foreign DNA or RNA by means of
infection by bacteria, molds, fungi, and viruses. Nucleic
acid hybridization has, until now, been employed primarily in
academic and industrial molecular biology laboratories. The
application of nucleic acid hybridization as a diagnostic tool
in clinical medicine is limited because of the frequently very
low concentrations of disease related DNA or RNA present in a
patient's body fluid and the unavailability of a sufficiently
sensitive method of nucleic acid hybridization analysis.
Current methods for detecting specific nucleic acid
sequences generally involve immobilization of the target
nucleic acid on a solid support such as nitrocellulose paper,
cellulose paper, diazotized paper, or a nylon membrane. After
the target nucleic acid is fixed on the support, the support
is contacted with a suitably labelled probe nucleic acid for
about two to forty-eight hours. After the above time period,
the solid support is washed several times at a controlled
temperature to remove unhybridized probe. The support is then
dried and the hybridized material is detected by
autoradiography or by spectrometric methods.
When very low concentrations must be detected, the
current methods are slow and labor intensive, and nonisotopic
labels that are less readily detected than radiolabels are
frequently not suitable. A method for increasing the
sensitivity to permit the use of simple, rapid, nonisotopic,
homogeneous or heterogeneous methods for detecting nucleic
acid sequences is therefore desirable.
Recently, a method for the enzymatic amplification of
specific segments of DNA known as the polymerase chain
reaction (PCR) method has been described. This in vitro
amplification procedure uses two or more different
oligonucleotide primers for different strands of the target
nucleic acid and is based on repeated cycles of denaturation,
oligonucleotide primer annealing, and primer extension by
thermophilic polymerase, resulting in the exponential increase
in copies of the region flanked by the primers. The different
PCR primers, which anneal to opposite strands of the DNA, are
positioned so that the polymerase catalyzed extension product
of one primer can serve as a template strand for the other
primer, leading to the accumulation of discrete fragments
whose length is defined by the distance between the 5'-ends of
the oligonucleotide primers.
Other methods for amplifying nucleic acids are single
primer amplification, ligase chain reaction (LCR), nucleic
acid sequence based amplification (NASBA) and the Q-beta-replicase
method. Regardless of the amplification used, the
amplified product must be detected.
Depending on which of the above amplification methods are
employed, the methods generally employ from seven to twelve or
more reagents. Furthermore, the above methods provide for
exponential amplification of a target or a reporter
oligonucleotide. Accordingly, it is necessary to rigorously
avoid contamination of assay solutions by the amplified
products to avoid false positives. Some of the above methods
require expensive thermal cycling instrumentation and
additional reagents and sample handling steps are needed for
detection of the amplified product.
Most assay methods that do not incorporate amplification
of a target DNA avoid the problem of contamination, but they
are not adequately sensitive or simple. Some of the methods
involve some type of size discrimination such as
electrophoresis, which adds to the complexity of the methods.
One method for detecting nucleic acids is to employ
nucleic acid probes. One method utilizing such probes is
described in U.S. Patent No. 4,868,104, the disclosure of
which is incorporated herein by reference. A nucleic acid
probe may be, or may be capable of being, labeled with a
reporter group or may be, or may be capable of becoming, bound
to a support.
Detection of signal depends upon the nature of the label
or reporter group. If the label or reporter group is an
enzyme, additional members of the signal producing system
include enzyme substrates and so forth. The product of the
enzyme reaction is preferably a luminescent product, or a
fluorescent or non-fluorescent dye, any of which can be
detected spectrophotometrically, or a product that can be
detected by other spectrometric or electrometric means. If
the label is a fluorescent molecule, the medium can be
irradiated and the fluorescence determined. Where the label
is a radioactive group, the medium can be counted to determine
the radioactive count.
It is desirable to have a sensitive, simple method for
detecting nucleic acids. The method should minimize the
number and complexity of steps and reagents. The need for
sterilization and other steps needed to prevent contamination
of assay mixtures should be avoided.
Description of the Related Art
Methods for detecting nucleic acid sequences are
discussed by Duck, et al., in U.S. Patent No. 5,011,769 and
corresponding International Patent Application WO 89/10415. A
method of cleaving a nucleic acid molecule is disclosed in
European Patent Application 0 601 834 A1 (Dahlberg, et al.).
Holland, et al., Clinical Chemistry (1992) 38:462-463,
describe detection of specific polymerase chain reaction
product by utilizing the 5' to 3' exonuclease activity of
Thermus aquaticus DNA polymerase. Longley, et al., Nucleic
Acids Research (1990) 18:7317-7322, discuss characterization
of the 5' to 3' exonuclease associated with Thermus aquaticus
DNA polymerase. Lyamichev, et al., Science (1993) 260:778-783,
disclose structure-specific endonucleolytic cleavage of
nucleic acids by eubacterial DNA polymerases.
A process for amplifying, detecting and/or cloning
nucleic acid sequences is disclosed in U.S. Patent Nos.
4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182.
Sequence polymerization by polymerase chain reaction is
described by Saiki, et al., (1986) Science, 230: 1350-1354.
Primer-directed enzymatic amplification of DNA with a
thermostable DNA polymerase is described by Saiki, et al.,
Science (1988) 239:487.
U.S. Patent Applications Serial Nos. 07/299,282 and
07/399,795, filed January 19, 1989, and August 29, 1989,
respectively, describe nucleic acid amplification using a
single polynucleotide primer. The disclosures of these
applications are incorporated herein by reference including
the references listed in the sections entitled "Description of
the Related Art."
Other methods of achieving the result of a nucleic acid
amplification are described by Van Brunt in Bio/Technology
(1990) 8(No.4): 291-294. These methods include ligase chain
reaction (LCR), nucleic acid sequence based amplification
(NASBA) and Q-beta-replicase amplification of RNA. LCR is
also discussed in European Patent Applications Nos. 439,182
(Backman I) and 473,155 (Backman II).
NASBA is a promoter-directed, isothermal enzymatic
process that induces in vitro continuous, homogeneous and
isothermal amplification of specific nucleic acid.
Q-beta-replicase relies on the ability of Q-beta-replicase
to amplify its RNA substrate exponentially under
isothermal conditions.
Another method for conducting an amplification of
nucleic acids is referred to as strand displacement
amplification (SDA). SDA is an isothermal, in vitro DNA
amplification technique based on the ability of a restriction
enzyme to nick the unmodified strand of a hemiphosphorothioate
form of its restriction site and the ability of a DNA
polymerase to initiate replication at the nick and displace
the downstream nontemplate strand intact. Primers containing
the recognition sites for the nicking restriction enzyme drive
the exponential amplification.
Another amplification procedure for amplifying nucleic
acids is known as 3SR, which is an RNA specific target method
whereby RNA is amplified in an isothermal process combining
promoter directed RNA polymerase, reverse transcriptase and
RNase H with target RNA.
SUMMARY OF THE INVENTION
The present invention is based on a method for modifying
an oligonucleotide. The method comprises incubating the
oligonucleotide with a polynucleotide and a 5'-nuclease
wherein at least a portion of the oligonucleotide is
reversibly hybridized to the polynucleotide under isothermal
conditions. The oligonucleotide is cleaved to provide (i) a
first fragment that is substantially non-hybridizable to the
polynucleotide and includes no more than one nucleotide from
the 5'-end of the portion and (ii) a second fragment that is
3' of the first fragment with reference to the intact
oligonucleotide and is substantially hybridizable to the
polynucleotide.
The above method has application to the detection of a
polynucleotide analyte. An oligonucleotide is reversibly
hybridized with a polynucleotide analyte and a 5'-nuclease
under isothermal conditions. The polynucleotide analyte
serves as a recognition element to enable a 5'-nuclease to
cleave the oligonucleotide to provide (i) a first fragment
that is substantially non-hybridizable to the polynucleotide
analyte and (ii) a second fragment that lies 3' of the first
fragment (in the intact oligonucleotide) and is substantially
hybridizable to the polynucleotide analyte. At least a 100-fold
molar excess of the first fragment and/or the second
fragment are obtained relative to the molar amount of the
polynucleotide analyte. The presence of the first fragment
and/or the second fragment is detected, the presence thereof
indicating the presence of the polynucleotide analyte.
With regard to a method for detecting a polynucleotide
analyte a combination is provided comprising a medium
suspected of containing the polynucleotide analyte, an excess,
relative to the suspected concentration of the polynucleotide
analyte, of a first oligonucleotide at least a portion of
which is capable of reversibly hybridizing with the
polynucleotide analyte under isothermal conditions, a 5'-nuclease,
and a second oligonucleotide having the
characteristic of hybridizing to a site on the polynucleotide
analyte that is 3' of the site at which the first
oligonucleotide hybridizes. The polynucleotide analyte is
substantially fully hybridized to the second oligonucleotide
under such isothermal conditions. The polynucleotide is
reversibly hybridized under the isothermal conditions to the
first oligonucleotide, which is cleaved as a function of the
presence of the polynucleotide analyte to provide, in at least
a 100-fold molar excess of the polynucleotide analyte, (i) a
first fragment that is substantially non-hybridizable to the
polynucleotide analyte and/or (ii) a second fragment that lies
3' of the first fragment (in the intact first oligonucleotide)
and is substantially hybridizable to the polynucleotide
analyte. The presence of the first fragment and/or the second
fragment is detected, the presence thereof indicating the
presence of the polynucleotide analyte.
The above methods have also application to the detection
of a DNA analyte. A combination is provided comprising a
medium suspected of containing the DNA analyte, a first
oligonucleotide at least a portion of which is capable of
reversibly hybridizing with the DNA analyte under isothermal
conditions, a 5'-nuclease, and a second oligonucleotide having
the characteristic of hybridizing to a site on the DNA analyte
that is 3' of the site at which the first oligonucleotide
hybridizes. The DNA analyte is substantially fully hybridized
to the second oligonucleotide under isothermal conditions.
The polynucleotide analyte is reversibly hybridized to the
first oligonucleotide under isothermal conditions. The first
oligonucleotide is cleaved to (i) a first fragment that is
substantially non-hybridizable to the DNA analyte and (ii) a
second fragment that lies 3' of the first fragment (in the
intact first oligonucleotide) and is substantially
hybridizable to the DNA analyte. At least a 100-fold molar
excess, relative to the DNA analyte, of the first fragment
and/or the second fragment is produced. The presence of the
first fragment and/or the second fragment is detected, the
presence thereof indicating the presence of the DNA analyte.
The present invention relates to a kit for detection of a
polynucleotide. The kit comprises in packaged combination (a)
a first oligonucleotide having the characteristic that, when
reversibly hybridized under isothermal conditions to the
polynucleotide, it is degraded by a 5'-nuclease to provide (i)
a first fragment that is substantially non-hybridizable to the
polynucleotide and (ii) a second fragment that is 3' of the
first fragment (in the first oligonucleotide) and is
substantially hybridizable to the polynucleotide, (b) a second
oligonucleotide having the characteristic of hybridizing to a
site on the polynucleotide that is separated by no more than
one nucleotide from the 3'-end of the site at which the first
oligonucleotide hybridizes wherein the polynucleotide is
substantially fully hybridized to the second oligonucleotide
under the isothermal conditions, and (c) a 5'-nuclease.
Brief Description of the Drawings
Figs. 1-3 are schematics of different embodiments in
accordance with the present invention.
Description of the Specific Embodiments
The present invention permits catalyzed cleavage of an
oligonucleotide that is modulated by a portion of a
polynucleotide analyte, such as a polynucleotide, that is
comprised of a target polynucleotide sequence to which a
portion of the oligonucleotide hybridizes. As such, the
methods of the present invention provide for very high
sensitivity assays for polynucleotide analytes. The methods
are simple to conduct and no temperature cycling is required.
Consequently, no expensive thermal cycling instrumentation is
needed. Furthermore, only a few reagents are used, thus
further minimizing cost and complexity of an assay. In
addition, the absence of amplified products, which are
potential amplification targets, permits the use of less
rigorous means to avoid contamination of assay solutions by
target sequences that could produce false positives.
Before proceeding further with a description of the
specific embodiments of the present invention, a number of
terms will be defined.
Polynucleotide analyte -- a compound or composition to be
measured that is a polymeric nucleotide, which in the intact
natural state can have about 20 to 500,000 or more nucleotides
and in an isolated state can have about 30 to 50,000 or more
nucleotides, usually about 100 to 20,000 nucleotides, more
frequently 500 to 10,000 nucleotides. Isolation of analytes
from the natural state, particularly those having a large
number of nucleotides, frequently results in fragmentation.
The polynucleotide analytes include nucleic acids from any
source in purified or unpurified form including DNA (dsDNA and
ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondrial
DNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or
mixtures thereof, genes, chromosomes, plasmids, the genomes of
biological material such as microorganisms, e.g., bacteria,
yeasts, viruses, viroids, molds, fungi, plants, animals,
humans, and fragments thereof, and the like. Preferred
polynucleotide analytes are double stranded DNA (dsDNA) and
single stranded DNA (ssDNA). The polynucleotide analyte can be
only a minor fraction of a complex mixture such as a
biological sample. The analyte can be obtained from various
biological material by procedures well known in the art. Some
examples of such biological material by way of illustration
and not limitation are disclosed in Table I below.
Microorganisms of interest include: |
Corynebacteria |
Corynebacterium diphtheria |
Pneumococci |
Diplococcus pneumoniae |
Streptococci |
Streptococcus pyrogenes |
Streptococcus salivarus |
Staphylococci |
Staphylococcus aureus |
Staphylococcus albus |
Neisseria |
Neisseria meningitidis |
Neisseria gonorrhea |
Enterobacteriaciae |
Escherichia coli |
Aerobacter aerogenes | The colliform |
Klebsiella pneumoniae | bacteria |
Salmonella typhosa |
Salmonella choleraesuis | The Salmonellae |
Salmonella typhimurium |
Shigella dysenteria |
Shigella schmitzii |
Shigella arabinotarda |
| The Shigellae |
Shigella flexneri |
Shigella boydii |
Shigella sonnei |
Other enteric bacilli |
Proteus vulgaris |
Proteus mirabilis | Proteus species |
Proteus morgani |
Pseudomonas aeruginosa |
Alcaligenes faecalis |
Vibrio cholerae |
Hemophilus-Bordetella group | Rhizopus oryzae |
Hemophilus influenza, H. ducryi | Rhizopus arrhizua |
Phycomycetes |
Hemophilus hemophilus | Rhizopus nigricans |
Hemophilus aegypticus | Sporotrichum schenkii |
Hemophilus parainfluenza | Flonsecaea pedrosoi |
Bordetella pertussis | Fonsecacea compact |
Pasteurellae | Fonsecacea dermatidis |
Pasteurella pestis | Cladosporium carrionii |
Pasteurella tulareusis | Phialophora verrucosa |
Brucellae | Aspergillus nidulans |
Brucella melitensis | Madurella mycetomi |
Brucella abortus | Madurella grisea |
Brucella suis | Allescheria boydii |
Aerobic Spore-forming Bacilli | Phialophora jeanselmei |
Bacillus anthracis | Microsporum gypseum |
Bacillus subtilis | Trichophyton mentagrophytes |
Bacillus megaterium | Keratinomyces ajelloi |
Bacillus cereus | Microsporum canis |
Anaerobic Spore-forming Bacilli | Trichophyton rubrum |
Clostridium botulinum | Microsporum adouini |
Clostridium tetani | Viruses |
Clostridium perfringens | Adenoviruses |
Clostridium novyi | Herpes Viruses |
Clostridium septicum | Herpes simplex |
Clostridium histolyticum | Varicella (Chicken pox) |
Clostridium tertium | Herpes Zoster (Shingles) |
Clostridium bifermentans | Virus B |
Clostridium sporogenes | Cytomegalovirus |
Mycobacteria | Pox Viruses |
Mycobacterium tuberculosis | Variola (smallpox) |
hominis |
Mycobacterium bovis | Vaccinia |
Mycobacterium avium | Poxvirus bovis |
Mycobacterium leprae | Paravaccinia |
Mycobacterium paratuberculosis | Molluscum contagiosum |
Actinomycetes (fungus-like bacteria) | Picornaviruses |
Actinomyces Isaeli | Poliovirus |
Actinomyces bovis | Coxsackievirus |
Actinomyces naeslundii | Echoviruses |
Nocardia asteroides | Rhinoviruses |
Nocardia brasiliensis | Myxoviruses |
The Spirochetes | Influenza(A, B, and C) |
Treponema pallidum Spirillum minus | Parainfluenza (1-4) |
Treponema pertenue Streptobacillus | Mumps Virus |
monoiliformis | Newcastle Disease Virus |
Treponema carateum | Measles Virus |
Borrelia recurrentis | Rinderpest Virus |
Leptospira icterohemorrhagiae | Canine Distemper Virus |
Leptospira canicola | Respiratory Syncytial Virus |
Trypanasomes | Rubella Virus |
Mycoplasmas | Arboviruses |
Mycoplasma pneumoniae |
Other pathogens | Eastern Equine Encephalitis |
Virus |
Listeria monocytogenes | Western Equine Encephalitis |
Virus |
Erysipelothrix rhusiopathiae | Sindbis Virus |
Streptobacillus moniliformis | Chikugunya Virus |
Donvania granulomatis | Semliki Forest Virus |
Bartonella bacilliformis | Mayora Virus |
Rickettsiae (bacteria-like parasites) | St. Louis Encephalitis Virus |
Rickettsia prowazekii | California Encephalitis Virus |
Rickettsia mooseri | Colorado Tick Fever Virus |
Rickettsia rickettsii | Yellow Fever Virus |
Rickettsia conori | Dengue Virus |
Rickettsia australis | Reoviruses |
Rickettsia sibiricus | Reovirus Types 1-3 |
| Retroviruses |
Rickettsia akari | Human Immunodeficiency Viruses (HIV) |
Rickettsia tsutsugamushi | Human T-cell Lymphotrophic Virus I & II (HTLV) |
Rickettsia burnetti | Hepatitis |
Rickettsia quintana | Hepatitis A Virus |
Chlamydia (unclassifiable parasites | Hepatitis B Virus |
bacterial/viral) | Hepatitis nonA-nonB Virus |
Chlamydia agents (naming uncertain) | Tumor Viruses |
Fungi | Rauscher Leukemia Virus |
Cryptococcus neoformans | Gross Virus |
Blastomyces dermatidis | Maloney Leukemia Virus |
Hisoplasma capsulatum |
Coccidioides immitis | Human Papilloma Virus |
Paracoccidioides brasiliensis |
Candida albicans |
Aspergillus fumigatus |
Mucor corymbifer (Absidia corymbifera) |
The polynucleotide analyte, where appropriate, may be
treated to cleave the analyte to obtain a polynucleotide that
contains a target polynucleotide sequence, for example, by
shearing or by treatment with a restriction endonuclease or
other site specific chemical cleavage method. However, it is
an advantage of the present invention that the polynucleotide
analyte can be used in its isolated state without further
cleavage.
For purposes of this invention, the polynucleotide
analyte, or a cleaved polynucleotide obtained from the
polynucleotide analyte, will usually be at least partially
denatured or single stranded or treated to render it denatured
or single stranded. Such treatments are well-known in the art
and include, for instance, heat or alkali treatment. For
example, double stranded DNA can be heated at 90-100° C. for a
period of about 1 to 10 minutes to produce denatured material.
3'- or 5'-End of an oligonucleotide -- as used herein
this phrase refers to a portion of an oligonucleotide
comprising the 3'- or 5'- terminus, respectively, of the
oligonucleotide.
3'- or 5'-Terminus of an oligonucleotide -- as used
herein this term refers to the terminal nucleotide at the 3'-
or 5'- end, respectively, of an oligonucleotide.
Target polynucleotide sequence -- a sequence of
nucleotides to be identified, which may be the polynucleotide
analyte but is usually existing within a polynucleotide
comprising the polynucleotide analyte. The identity of the
target polynucleotide sequence is known to an extent
sufficient to allow preparation of an oligonucleotide having a
portion or sequence that hybridizes with the target
polynucleotide sequence. In general, when one oligonucleotide
is used, the oligonucleotide hybridizes with the 5'-end of the
target polynucleotide sequence. When a second oligonucleotide
is used, it hybridizes to a site on the target polynucleotide
sequence that is 3' of the site to which the first
oligonucleotide hybridizes. (It should be noted that the
relationship can be considered with respect to the double
stranded molecule formed when the first and second
oligonucleotides are hybridized to the polynucleotide. In
such context the second oligonucleotide is 5-primeward of the
first oligonucleotide with respect to the "strand" comprising
the first and second oligonucleotides.) The relationships
described above are more clearly seen with reference to Fig.
3. The target polynucleotide sequence usually contains from
about 10 to 1,000 nucleotides, preferably 15 to 100
nucleotides, more preferably, 20 to 70 nucleotides. The
target polynucleotide sequence is part of a polynucleotide
that may be the entire polynucleotide analyte. The minimum
number of nucleotides in the target polynucleotide sequence is
selected to assure that the presence of target polynucleotide
sequence in a sample is a specific indicator of the presence
of polynucleotide analyte in a sample. Very roughly, the
sequence length is usually greater than about 1.6 log L
nucleotides where L is the number of base pairs in the genome
of the biologic source of the sample. The number of
nucleotides in the target sequence is usually the sum of the
lengths of those portions of the oligonucleotides that
hybridize with the target sequence plus the number of
nucleotides lying between the portions of the target sequence
that hybridize with the oligonucleotides.
Oligonucleotide -- a polynucleotide, usually a synthetic
polynucleotide, usually single stranded that is constructed
such that at least a portion thereof hybridizes with the
target polynucleotide sequence of the polynucleotide. The
oligonucleotides of this invention are usually 10 to 150
nucleotides, preferably, deoxyoligonucleotides of 15 to 100
nucleotides, more preferably, 20 to 60 nucleotides, in length.
The first oligonucleotide, or "the" oligonucleotide when
a second oligonucleotide is not employed, has a 5'-end about 0
to 100 nucleotides, preferably, 1 to 20 nucleotides in length
that does not hybridize with the target polynucleotide
sequence and usually has a 10 to 40 nucleotide sequence that
hybridizes with the target polynucleotide sequence. In
general, the degree of amplification is reduced somewhat as
the length of the portion of the oligonucleotide that does not
hybridize with the target polynucleotide sequence increases.
The first oligonucleotide also may have a sequence at its 3'-end
that does not hybridize with the target polynucleotide
sequence.
The second oligonucleotide preferably hybridizes at its
3'-end with the target polynucleotide sequence at a site on
the target polynucleotide sequence 3' of the site of binding
of the first oligonucleotide. The length of the portion of
the second oligonucleotide that hybridizes with the target
polynucleotide sequence is usually longer than the length of
the portion of the first oligonucleotide that hybridizes with
the target polynucleotide sequence and is usually 20 to 100
nucleotides. The melting temperature of the second
oligonucleotide hybridized to the target polynucleotide
sequence is preferably at least as high, more preferably, at
least 5°C higher than the melting temperature of the first
oligonucleotide hybridized to the target polynucleotide
sequence.
The oligonucleotides can be oligonucleotide mimics such a
polynucleopeptides, phosphorothioates or phosphonates except
that the first oligonucleotide usually has at least one
phosphodiester bond to the nucleoside at the 5'-end of the
sequence that hybridizes with the target polynucleotide
sequence. When oligonucleotide mimics are used that provide
very strong binding, such as polynucleopeptides, the length of
the portion of the second oligonucleotide that hybridizes with
the target polynucleotide sequence may be reduced to less than
20 and, preferably, greater than 10.
Various techniques can be employed for preparing an
oligonucleotide or other polynucleotide utilized in the
present invention. They can be obtained by biological
synthesis or by chemical synthesis. For short
oligonucleotides (up to about 100 nucleotides) chemical
synthesis will frequently be more economical as compared to
biological synthesis. In addition to economy, chemical
synthesis provides a convenient way of incorporating low
molecular weight compounds and/or modified bases during the
synthesis step. Furthermore, chemical synthesis is very
flexible in the choice of length and region of the target
polynucleotide sequence. The oligonucleotides can be
synthesized by standard methods such as those used in
commercial automated nucleic acid synthesizers. Chemical
synthesis of DNA on a suitably modified glass or resin results
in DNA covalently attached to the surface. This may offer
advantages in washing and sample handling. For longer
sequences standard replication methods employed in molecular
biology can be used such as the use of M13 for single stranded
DNA as described by J. Messing (1983) Methods Enzymol, 101,
20-78.
In addition to standard cloning techniques, in vitro
enzymatic methods may be used such as polymerase catalyzed
reactions. For preparation of RNA, T7 RNA polymerase and a
suitable DNA template can be used. For DNA, polymerase chain
reaction (PCR) and single primer amplification are convenient.
Other chemical methods of polynucleotide or
oligonucleotide synthesis include phosphotriester and
phosphodiester methods (Narang, et al., Meth. Enzymol (1979)
68: 90) and synthesis on a support (Beaucage, et al.,
Tetrahedron (1981) Letters 22: 1859-1862) as well as
phosphoramidate techniques, Caruthers, M. H., et al., "Methods
in Enzymology," Vol. 154, pp. 287-314 (1988), and others
described in "Synthesis and Applications of DNA and RNA," S.A.
Narang, editor, Academic Press, New York, 1987, and the
references contained therein.
Fragment -- in general, in the present method the
oligonucleotide (or the first oligonucleotide when a second
oligonucleotide is employed) is cleaved only when at least a
portion thereof is reversibly hybridized with a target
polynucleotide sequence and, thus, the target polynucleotide
sequence acts as a recognition element for cleavage of the
oligonucleotide, thereby yielding two portions. One fragment
is substantially non-hybridizable to the target polynucleotide
sequence. The other fragment is substantially hybridizable to
the target polynucleotide sequence and 3' of the other
fragment with respect to the oligonucleotide in its uncleaved
form.
5'-Nuclease -- a sequence-independent deoxyribonuclease
enzyme that catalyzes the cleavage of an oligonucleotide into
fragments only when at least a portion of the oligonucleotide
is hybridized to the target polynucleotide sequence. The
enzyme selectively cleaves the oligonucleotide near the 5'-terminus
of the bound portion, within 5 nucleotides thereof,
preferably within 1 to 2 nucleotides thereof and does not
cleave the unhybridized oligonucleotide or the target
polynucleotide sequence. Such enzymes include both 5'-exonucleases
and 5'-endonucleases but exclude ribonucleases
such as RNAse H and restriction enzymes. 5'-nucleases useful
in the present invention must be stable under the isothermal
conditions used in the present method and are usually
thermally stable nucleotide polymerases having 5'-exonuclease
activity such as Taq DNA polymerase (e.g. AmpliTaq(TM) from
Perkin-Elmer Corporation, Norwalk, N.J.), Thermalase Tbr(TM)
DNA polymerase (from Amresco, Solon, Ohio), Ultra Therm(TM)
DNA polymerase (from Bio/Can Scientific, Ontario, Canada),
Replitherm(TM) DNA polymerase (from Epicentre, Madison,
Wisconsin), Tfl(TM) DNA polymerase (from Epicentre),
Panozyme(TM) DNA polymerase (from Panorama Research, Mountain
View, California), Tth(TM) DNA polymerase (from Epicentre),
rBst(TM) DNA polymerase (from Epicentre), Heat Tuff(TM) DNA
polymerase (from Clontech, Palo Alto, California), and the
like, derived from any source such as cells, bacteria, such as
E. coli, plants, animals, virus, thermophilic bacteria, and so
forth wherein the polymerase may be modified chemically or
through genetic engineering to provide for thermal stability
and/or increased activity.
Isothermal conditions -- a uniform or constant
temperature at which the modification of the oligonucleotide
in accordance with the present invention is carried out. The
temperature is chosen so that the duplex formed by hybridizing
the oligonucleotide to a polynucleotide with a target
polynucleotide sequence is in equilibrium with the free or
unhybridized oligonucleotide and free or unhybridized target
polynucleotide sequence, a condition that is otherwise
referred to herein as "reversibly hybridizing" the
oligonucleotide with a polynucleotide. Normally, at least 1%,
preferably 20 to 80%, usually less than 95% of the
polynucleotide is hybridized to the oligonucleotide under the
isotermal conditions. Accordingly, under isothermal
conditions there are molecules of polynucleotide that are
hybridized with the oligonucleotide, or portions thereof, and
are in dynamic equilibrium with molecules that are not
hybridized with the oligonucleotide. Some fluctuation of the
temperature may occur and still achieve the benefits of the
present invention. The fluctuation generally is not necessary
for carrying out the methods of the present invention and
usually offer no substantial improvement. Accordingly, the
term "isothermal conditions" includes the use of a fluctuating
temperature, particularly random or uncontrolled fluctuations
in temperature, but specifically excludes the type of
fluctuation in temperature referred to as thermal cycling,
which is employed in some known amplification procedures,
e.g., polymerase chain reaction.
Polynucleotide primer(s) or oligonucleotide
primer(s) -- an oligonucleotide that is usually employed in a
chain extension on a polynucleotide template.
Nucleoside triphosphates -- nucleosides having a
5'-triphosphate substituent. The nucleosides are pentose
sugar derivatives of nitrogenous bases of either purine or
pyrimidine derivation, covalently bonded to the 1'-carbon of
the pentose sugar, which is usually a deoxyribose or a ribose.
The purine bases include adenine(A), guanine(G), inosine, and
derivatives and analogs thereof. The pyrimidine bases include
cytosine (C), thymine (T), uracil (U), and derivatives and
analogs thereof. Nucleoside triphosphates include
deoxyribonucleoside triphosphates such as dATP, dCTP, dGTP and
dTTP and ribonucleoside triphosphates such as rATP, rCTP, rGTP
and rUTP. The term "nucleoside triphosphates" also includes
derivatives and analogs thereof.
Nucleotide -- a base-sugar-phosphate combination that is
the monomeric unit of nucleic acid polymers, i.e., DNA and
RNA.
Nucleoside -- is a base-sugar combination or a nucleotide
lacking a phosphate moiety.
Nucleotide polymerase -- a catalyst, usually an enzyme,
for forming an extension of an oligonucleotide along a
polynucleotide template where the extension is complementary
thereto. The nucleotide polymerase is a template dependent
polynucleotide polymerase and utilizes nucleoside
triphosphates as building blocks for extending the 3'-end of a
oligonucleotide to provide a sequence complementary with the
single stranded portion of the polynucleotide to which the
oligonucleotide is hybridized to form a duplex.
Hybridization (hybridizing) and binding -- in the context
of nucleotide sequences these terms are used interchangeably
herein. The ability of two nucleotide sequences to hybridize
with each other is based on the degree of complementarity of
the two nucleotide sequences, which in turn is based on the
fraction of matched complementary nucleotide pairs. The more
nucleotides in a given sequence that are complementary to
another sequence, the more stringent the conditions can be for
hybridization and the more specific will be the binding of the
two sequences. Increased stringency is achieved by elevating
the temperature, increasing the ratio of cosolvents, lowering
the salt concentration, and the like.
Homologous or substantially identical -- In general, two
polynucleotide sequences that are identical or can each
hybridize to the same polynucleotide sequence are homologous.
The two sequences are homologous or substantially identical
where the sequences each have at least 90%, preferably 100%,
of the same or analogous base sequence where thymine (T) and
uracil (U) are considered the same. Thus, the ribonucleotides
A, U, C and G are taken as analogous to the deoxynucleotides
dA, dT, dC, and dG, respectively. Homologous sequences can
both be DNA or one can be DNA and the other RNA.
Complementary -- Two sequences are complementary when the
sequence of one can bind to the sequence of the other in an
anti-parallel sense wherein the 3'-end of each sequence binds
to the 5'-end of the other sequence and each A, T(U), G, and C
of one sequence is then aligned with a T(U), A, C, and G,
respectively, of the other sequence.
Copy -- means a sequence that is a direct identical or
homologous copy of a single stranded polynucleotide sequence
as differentiated from a sequence that is complementary to the
sequence of such single stranded polynucleotide.
Member of a specific binding pair ("sbp member") -- one
of two different molecules, having an area on the surface or
in a cavity which specifically binds to, and is thereby
defined as complementary with, a particular spatial and polar
organization of the other molecule. The members of the
specific binding pair are referred to as ligand and receptor
(antiligand). These may be members of an immunological pair
such as antigen-antibody, or may be operator-repressor,
nuclease-nucleotide, biotin-avidin, hormones-hormone
receptors, nucleic acid duplexes, IgG-protein A, DNA-DNA,
DNA-RNA, and the like.
Ligand -- any compound for which a receptor naturally
exists or can be prepared.
Receptor ("antiligand") -- any compound or composition
capable of recognizing a particular spatial and polar
organization of a molecule, e.g., epitopic or determinant
site. Illustrative receptors include naturally occurring
receptors, e.g., thyroxine binding globulin, antibodies,
enzymes, Fab fragments, lectins, nucleic acids, repressors,
protection enzymes, protein A, complement component Clq, DNA
binding proteins or ligands and the like.
Small organic molecule -- a compound of molecular weight
less than 1500, preferably 100 to 1000, more preferably 300 to
600 such as biotin, fluorescein, rhodamine and other dyes,
tetracycline and other protein binding molecules, and haptens,
etc. The small organic molecule can provide a means for
attachment of a nucleotide sequence to a label or to a support
or may itself be a label.
Support or surface -- a porous or non-porous water
insoluble material. The support can be hydrophilic or capable
of being rendered hydrophilic and includes inorganic powders
such as silica, magnesium sulfate, and alumina; natural
polymeric materials, particularly cellulosic materials and
materials derived from cellulose, such as fiber containing
papers, e.g., filter paper, chromatographic paper, etc.;
synthetic or modified naturally occurring polymers, such as
nitrocellulose, cellulose acetate, poly (vinyl chloride),
polyacrylamide, cross linked dextran, agarose, polyacrylate,
polyethylene, polypropylene, poly(4-methylbutene),
polystyrene, polymethacrylate, poly(ethylene terephthalate),
nylon, poly(vinyl butyrate), etc.; either used by themselves
or in conjunction with other materials; glass available as
Bioglass, ceramics, metals, and the like. Natural or
synthetic assemblies such as liposomes, phospholipid vesicles,
and cells can also be employed.
Binding of sbp members to a support or surface may be
accomplished by well-known techniques, commonly available in
the literature. See, for example, "Immobilized Enzymes,"
Ichiro Chibata, Halsted Press, New York (1978) and
Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface can
have any one of a number of shapes, such as strip, rod,
particle, including bead, and the like.
Label or reporter group or reporter molecule -- a member
of a signal producing system. Usually the label or reporter
group or reporter molecule is conjugated to or becomes bound
to, or fragmented from, an oligonucleotide or to a nucleoside
triphosphate and is capable of being detected directly or,
through a specific binding reaction, and can produce a
detectible signal. In general, any label that is detectable
can be used. The label can be isotopic or nonisotopic,
usually non-isotopic, and can be a catalyst, such as an enzyme
or a catalytic polynucleotide, promoter, dye, fluorescent
molecule, chemiluminescer, coenzyme, enzyme substrate,
radioactive group, a small organic molecule, amplifiable
polynucleotide sequence, a particle such as latex or carbon
particle, metal sol, crystallite, liposome, cell, etc., which
may or may not be further labeled with a dye, catalyst or
other detectible group, and the like. Labels include an
oligonucleotide or specific polynucleotide sequence that can
provide a template for amplification or ligation or act as a
ligand such as for a repressor protein. The label is a member
of a signal producing system and can generate a detectable
signal either alone or together with other members of the
signal producing system. The label can be bound directly to a
nucleotide sequence or can become bound thereto by being bound
to an sbp member complementary to an sbp member that is bound
to a nucleotide sequence.
Signal Producing System -- The signal producing system
may have one or more components, at least one component being
the label or reporter group or reporter molecule. The signal
producing system generates a signal that relates to the
presence or amount of target polynucleotide sequence or a
polynucleotide analyte in a sample. The signal producing
system includes all of the reagents required to produce a
measurable signal. When the label is not conjugated to a
nucleotide sequence, the label is normally bound to an sbp
member complementary to an sbp member that is bound to, or
part of, a nucleotide sequence. Other components of the
signal producing system may be included in a developer
solution and can include substrates, enhancers, activators,
chemiluminescent compounds, cofactors, inhibitors, scavengers,
metal ions, specific binding substances required for binding
of signal generating substances, and the like. Other
components of the signal producing system may be coenzymes,
substances that react with enzymic products, other enzymes and
catalysts, and the like. The signal producing system provides
a signal detectable by external means, by use of
electromagnetic radiation, desirably by visual examination.
The signal-producing system is described more fully in U.S.
Patent Application Serial No. 07/555,323, filed July 19, 1990,
the relevant disclosure of which is incorporated herein by
reference.
Amplification of nucleic acids or polynucleotides -- any
method that results in the formation of one or more copies of
a nucleic acid or a polynucleotide molecule, usually a nucleic
acid or polynucleotide analyte, or complements thereof,
present in a medium.
Exponential amplification of nucleic acids or
polynucleotides -- any method that results in the formation of
one or more copies of a nucleic acid or polynucleotide
molecule, usually a nucleic acid or polynucleotide analyte,
present in a medium.
Methods for the enzymatic amplification of specific
double stranded sequences of DNA include those described above
such as the polymerase chain reaction (PCR), amplification of
a single stranded polynucleotide using a single polynucleotide
primer, ligase chain reaction (LCR), nucleic acid sequence
based amplification (NASBA), Q-beta-replicase method, strand
displacement amplification (SDA), and 3SR.
Conditions for carrying out an amplification, thus, vary
depending upon which method is selected. Some of the methods
such as PCR utilize temperature cycling to achieve
denaturation of duplexes, oligonucleotide primer annealing,
and primer extension by thermophilic template dependent
polynucleotide polymerase. Other methods such as NASBA,
Q-beta-replicase method, SDA and 3SR are isothermal. As can
be seen, there are a variety of known amplification methods
and a variety of conditions under which these methods are
conducted to achieve exponential amplification.
Linear amplification of nucleic acids or polynucleotides
-- any method that results in the formation of one or more
copies of only the complement of a nucleic acid or
polynucleotide molecule, usually a nucleic acid or
polynucleotide analyte, present in a medium. Thus, one
difference between linear amplification and exponential
amplification is that the latter produces copies of the
polynucleotide whereas the former produces only the
complementary strand of the polynucleotide. In linear
amplification the number of complements formed is, in
principle, directly proportional to the time of the reaction
as opposed to exponential amplification wherein the number of
copies is, in principle, an exponential function of the time
or the number of temperature cycles.
Ancillary Materials -- various ancillary materials will
frequently be employed in the methods and assays carried out
in accordance with the present invention. For example,
buffers will normally be present in the assay medium, as well
as stabilizers for the assay medium and the assay components.
Frequently, in addition to these additives, proteins may be
included, such as albumins, organic solvents such as
formamide, quaternary ammonium salts, polycations such as
dextran sulfate, surfactants, particularly non-ionic
surfactants, binding enhancers, e.g., polyalkylene glycols, or
the like.
As mentioned above, the present invention has a primary
application to methods for detecting a polynucleotide analyte.
In one aspect of the invention an oligonucleotide is
reversibly hybridized with a polynucleotide analyte in the
presence of a 5'-nuclease under isothermal conditions. In
this way the polynucleotide analyte serves as a "recognition
element" to enable the 5'-nuclease to specifically cleave the
oligonucleotide to provide first and second fragments when the
oligonucleotide is reversibly hybridized to the polynucleotide
analyte. The first fragment comprises the 5'-end of the
oligonucleotide (with reference to the intact or original
oligonucleotide) and is substantially non-hybridizable to the
polynucleotide analyte and can serve as a label. The first
fragment generally includes at least a portion of that part
the 5'-end of the original oligonucleotide that was not
hybridized to the polynucleotide analyte when the portion of
the oligonucleotide that is hybridizable with the
polynucleotide analyte is reversibly hybridized thereto.
Additionally, the first fragment may include nucleotides
(usually, no more than 5, preferably, no more than 2, more
preferably, no more than 1 of such nucleotides) that are
cleaved by the 5'-nuclease from the 5'-end of that portion (or
sequence) of the original oligonucleotide that was hybridized
to the polynucleotide analyte. Therefore, it is in the above
context that the first fragment is "substantially non-hybridizable"
with the polynucleotide analyte. The second
fragment comprises the sequence of nucleotides at the 3'-end
of the oligonucleotide that were reversibly hybridized to the
polynucleotide analyte minus those nucleotides cleaved by the
5'-nuclease when the original oligonucleotide is reversibly
hybridized to the polynucleotide analyte. Accordingly, the
second fragment is "substantially hybridizable" to the
polynucleotide analyte having resulted from that portion of
the oligonucleotide that reversibly hybridizes with the
polynucleotide analyte.
As mentioned above, the 3'-end of the oligonucleotide may
include one or more nucleotides that do not hybridize with the
polynucleotide analyte and may comprise a label. At least a
100-fold molar excess of the first fragment and/or the second
fragment are obtained relative to the molar amount of the
polynucleotide analyte. The sequence of at least one of the
fragments is substantially preserved during the reaction. The
presence of the first fragment and/or the second fragment is
detected, the presence thereof indicating the presence of the
polynucleotide analyte.
The 5'-nuclease is generally present in an amount
sufficient to cause the cleavage of the oligonucleotide, when
it is reversibly hybridized to the polynucleotide analyte, to
proceed at least half as rapidly as the maximum rate
achievable with excess enzyme, preferably, at least 75% of the
maximum rate. The concentration of the
5'-nuclease is usually determined empirically. Preferably, a
concentration is used that is sufficient such that further
increase in the concentration does not decrease the time for
the amplification by over 5-fold, preferably 2-fold. The
primary limiting factor generally is the cost of the reagent.
In this respect, then, the polynucleotide analyte, or at least
the target polynucleotide sequence, and the enzyme are
generally present in a catalytic amount. The
oligonucleotide that is cleaved by the enzyme is usually in
large excess, preferably, 10-9 M to 10-5 M, and is used in an
amount that maximizes the overall rate of its cleavage in
accordance with the present invention wherein the rate is at
least 10%, preferably, 50%, more preferably, 90%, of the
maximum rate of reaction possible. Concentrations of the
oligonucleotide lower than 50% may be employed to facilitate
detection of the fragment(s) produced in accordance with the
present invention. The amount of oligonucleotide is at least
as great as the number of molecules of product desired.
Usually, the concentration of the oligonucleotide is 0.1
nanomolar to 1 millimolar, preferably, 1 nanomolar to 10
micromolar. It should be noted that increasing the
concentration of the oligonucleotide causes the reaction rate
to approach a limiting value that depends on the
oligonucleotide sequence, the temperature, the concentration
of the target polynucleotide sequence and the enzyme
concentration. For many detection methods very high
concentrations of the oligonucleotide may make detection more
difficult.
The amount of the target polynucleotide sequence that is
to be copied can be as low as one or two molecules in a sample
but generally may vary from about 102 to 1010, more usually
from about 103 to 108 molecules in a sample preferably at least
10-21M in the sample and may be 10-10 to 10-19M, more usually 10-14
to 10-19M.
In carrying out the methods in accordance with the
present invention, an aqueous medium is employed. Other polar
solvents may also be employed as cosolvents, usually
oxygenated organic solvents of from 1-6, more usually from
1-4, carbon atoms, including alcohols, ethers and the like.
Usually these cosolvents, if used, are present in less than
about 70 weight percent, more usually in less than about 30
weight percent.
The pH for the medium is usually in the range of about
4.5 to 9.5, more usually in the range of about 5.5 - 8.5, and
preferably in the range of about 6 - 8. The pH and
temperature are chosen so as to achieve the reversible
hybridization or equilibrium state under which cleavage of an
oligonucleotide occurs in accordance with the present
invention. In some instances, a compromise is made in the
reaction parameters in order to optimize the speed,
efficiency, and specificity of these steps of the present
method. Various buffers may be used to achieve the desired pH
and maintain the pH during the determination. Illustrative
buffers include borate, phosphate, carbonate, Tris, barbital
and the like. The particular buffer employed is not critical
to this invention but in individual methods one buffer may be
preferred over another.
As mentioned above the reaction in accordance with the
present invention is carried out under isothermal conditions.
The reaction is generally carried out at a temperature that is
near the melting temperature of the
oligonucleotide:polynucleotide analyte complex. Accordingly,
the temperature employed depends on a number of factors.
Usually, for cleavage of the oligonucleotide in accordance
with the present invention, the temperature is about 35°C to
90°C depending on the length and sequence of the
oligonucleotide. It will usually be desired to use relatively
high temperature of 60°C to 85°C to provide for a high rate of
reaction. The amount of the fragments formed depends on the
incubation time and temperature. In general, a moderate
temperature is normally employed for carrying out the methods.
The exact temperature utilized also varies depending on the
salt concentration, pH, solvents used, and the length of and
composition of the target polynucleotide sequence as well as
the oligonucleotide as mentioned above.
One embodiment of the invention is depicted in Fig. 1.
Oligonucleotide OL is combined with polynucleotide analyte PA
having target polynucleotide sequence TPS and with a 5'-nuclease,
which can be, for example, a Taq polymerase. In
this embodiment OL is labeled (*) within what is designated
the first fragment, produced upon cleavage of the
oligonucleotide in accordance with the present invention. OL
in this embodiment usually is at least 10 nucleotides in
length, preferably, about 10 to 50 nucleotides in length, more
preferably; 15 to 30 or more nucleotides in length. In
general, the length of OL should be sufficient so that a
portion hybridizes with TPS, the length of such portion
approximating the length of TPS. In this embodiment the
length of OL is chosen so that the cleavage of no more than 5,
preferably, no more than 1 to 3, more preferably, 1 to 2
nucleotides, therefrom results in two fragments. The first
fragment, designated LN, is no more than 5 nucleotides in
length, preferably, 1 to 3 nucleotides in length, more
preferably, 1 to 2 nucleotides in length and the second
fragment, designated DOL, is no more than 5, preferably, no
more than 1 to 3, more preferably, no more than 1 to 2,
nucleotides shorter than the length of OL.
As shown in Fig. 1, OL hybridizes with TPS to give duplex
I. The hybridization is carried out under isothermal
conditions so that OL is reversibly hybridized with TPS. OL
in duplex I is cleaved to give DOL and LN, wherein LN includes
a labeled nucleotide (*). In the embodiment depicted in Fig.
1, DOL is the complement of TPS except for the nucleotides
missing at the 5'-end. Since during the course of the
isothermal reaction the 5'-end of PA may be cleaved at or near
the 5'-end of TPS, DOL may also have 0 to 5 nucleotides at its
3'-end that overhang and cannot hybridize with the residual
portion of TPS. The isothermal conditions are chosen such
that equilibrium exists between duplex I and its single
stranded components, namely, PA and OL. Upon cleavage of OL
within duplex I, an equilibrium is also established between
duplex I and its single stranded components, PA and DOL.
Since OL is normally present in large excess relative to the
amount of DOL formed in the reaction, there are usually many
more duplexes containing OL than DOL. The reaction described
above for duplex I continuously produces additional molecules
of DOL.
The reaction is allowed to continue until a sufficient
number of molecules of DOL and LN are formed to permit
detection of the labeled LN (LN*) and, thus, the
polynucleotide analyte. In this way the enzyme-catalyzed
cleavage of nucleotides from the 5'-end of OL is modulated by
and, therefore, related to the presence of the polynucleotide
analyte. Depending on the amount of PA present, a sufficient
number of molecules for detection can be obtained where the
time of reaction is from about 1 minute to 24 hours.
Preferably, the reaction can be carried out in less than 5
hours. As a matter of convenience it is usually desirable to
minimize the time period as long as the requisite of number of
molecules of detectable fragment is achieved. In general, the
time period for a given degree of cleavage can be minimized by
optimizing the temperature of the reaction and using
concentrations of the 5'-nuclease and the oligonucleotide that
provide reaction rates near the maximum achievable with excess
of these reagents. Detection of the polynucleotide analyte is
accomplished indirectly by detecting the label in fragment
LN*. Alternatively, DOL may be detected, for example, by
using the label as a means of separating LN* and OL from the
reaction mixture and then detecting the residual DOL.
Detection of the labeled fragment is facilitated in a
number of ways. For example, a specific pair member such as
biotin or a directly detectable label such a fluorescein can
be used. The low molecular weight LN* can be separated by
electrophoresis, gel exclusion chromatography, thin layer
chromatography ultrafiltration and the like and detected by
any convenient means such as a competitive binding assay or
direct detection of the label. Alternatively, the
oligonucleotide can be labeled within the second (DOL)
fragment with a specific binding member such as a ligand, a
small organic molecule, a polynucleotide sequence or a
protein, or with a directly detectable label such as a
directly detectable small organic molecules, e.g.,
fluorescein, a sensitizer, a coenzyme and the like. Detection
will then depend on differentiating the oligonucleotide with
labels on both ends from singly labeled fragments where one
labeled end has been cleaved. In this case it is desirable to
label one end of OL with a specific binding member that
facilitates removal of OL and the fragment retaining the label
by using a complementary sbp member bound to a support. The
residual labeled fragments bearing the other label are then
detected by using a method appropriate for detecting that
label.
One method for detecting nucleic acids is to employ
nucleic acid probes. Other assay formats and detection
formats are disclosed in U.S. Patent Applications Serial Nos.
07/229,282 and 07/399,795 filed January 19, 1989, and August
29, 1989, respectively, U.S. Patent Application Serial No.
07/555,323 filed July 19, 1990, U.S. Patent Application Serial
No. 07/555,968 and U.S. Patent Application Serial No.
07/776,538 filed October 11, 1991, which have been
incorporated herein by reference.
Examples of particular labels or reporter molecules and
their detection can be found in U.S. Patent Application Serial
No. 07/555,323 filed July 19, 1990, the relevant disclosure of
which is incorporated herein by reference.
Detection of the signal will depend upon the nature of
the signal producing system utilized. If the label or
reporter group is an enzyme, additional members of the signal
producing system include enzyme substrates and so forth. The
product of the enzyme reaction is preferably a luminescent
product, or a fluorescent or non-fluorescent dye, any of which
can be detected spectrophotometrically, or a product that can
be detected by other spectrometric or electrometric means. If
the label is a fluorescent molecule, the medium can be
irradiated and the fluorescence determined. Where the label
is a radioactive group, the medium can be counted to determine
the radioactive count.
Another embodiment of the present invention is depicted
in Fig. 2. Oligonucleotide OL' has a first portion or
sequence SOL1 that is not hybridized to TPS' and a second
portion or sequence SOL2 that is hybridized to TPS'. OL' is
combined with polynucleotide analyte PA' having target
polynucleotide sequence TPS' and with a 5'-endonuclease (5'-endo),
which can be, for example, Taq DNA polymerase and the
like. OL' and 5'-endo are generally present in concentrations
as described above. In the embodiment of Fig. 2, OL' is
labeled (*) within the sequence SOL1 wherein SOL1 may
intrinsically comprise the label or may be extrinsically
labeled with a specific binding member or directly detectable
labeled. The length of SOL2 is as described in the embodiment
of Fig. 1. In general, the length of SOL2 should be
sufficient to hybridize with TPS', usually approximating the
length of TPS'. SOL1 may be any length as long as it does not
substantially interfere with the cleavage of OL' and will
preferably be relatively short to avoid such interference.
Usually, SOL1 is about 1 to 100 nucleotides in length,
preferably, 8 to 20 nucleotides in length.
In this embodiment the cleavage of SOL1 from SOL2 results
in two fragments. Cleavage in SOL2 occurs within 5
nucleotides of the bond joining SOL1 and SOL2 in OL'. The
exact location of cleavage is not critical so long as the
enzyme cleaves OL' only when it is bound to TPS'. The two
fragments are designated LNSOL1 and DSOL2. LNSOL1 is
comprised of the 5'-end of OL' and DSOL2 is comprised of the
3'-end of OL'. The sequence of at least one of LNSOL1 and
DSOL2 remains substantially intact during the cleavage
reaction. As shown in Fig. 2, SOL2 of OL' hybridizes with
TPS' to give duplex I'. The hybridization is carried out
under isothermal conditions so that OL' is reversibly
hybridized with TPS'. OL' in duplex I' is cleaved to give
DSOL2 and LNSOL1, the latter of which comprises a label. In
the embodiment depicted in Fig. 2, DSOL2 is the complement of
TPS' except for any nucleotides missing at the 5'-end thereof
as a result of the cleavage of the cleavage reaction and any
nucleotides appended to the
3'-end of OL' (not shown in Fig. 2) that do not hybridize with
TPS'.
The isothermal conditions are chosen such that
equilibrium exists between duplex I' and its single stranded
components, i.e., PA' and OL'. Upon cleavage of OL' within
duplex I' and equilibrium is also established between duplex
I' and its single stranded components, PA' and DSOL2. Since
OL' is normally present in large excess relative to the amount
of DSOL2 formed in the reaction, there are usually many more
duplexes containing OL' than DSOL2. The reaction described
above for duplex I' continuously produces molecules of DSOL2
and LNSOL1. The reaction is allowed to continue until a
sufficient number of molecules of DSOL2 and LNSOL1 are formed
to permit detection of one or both of these fragments. In
this way the enzyme-catalyzed cleavage of LNSOL1 from the 5'-end
of the portion of OL' hybridized to PA' is modulated by,
and therefore related to, the presence of the polynucleotide
analyte. The reaction parameters and the detection of DSOL2
and/or LNSOL1 are generally as described above for the
embodiment of Fig.1.
Various ways of controlling the cleavage of the
oligonucleotide can be employed. For example, the point of
cleavage can be controlled by introducing a small organic
group, such as biotin, into the nucleotide at the
5'-terminus of OL' or the nucleotide in SOL2 that is at the
junction of SOL2 and SOL1.
An embodiment using a second oligonucleotide is depicted
in Fig. 3. The second oligonucleotide (OL2) hybridizes to a
site TPS2 on PA'' that lies 3' of the site of hybridization
(TPS1) of the sequence SOL2" of the first oligonucleotide,
namely, OL''. In the embodiment shown OL2 fully hybridizes
with TPS2. This is by way of example and not limitation. The
second oligonucleotide can include nucleotides at its 5'end
that are not hybridizable with the target polynucleotide
sequence, but its 3'-end is preferably hybridizable.
Preferably, OL2 binds to a site (TPS2) that is contiguous with
the site to which SOL2'' hybridizes (TPS1). However, it is
within the purview of the present invention that the second
oligonucleotide hybridize with PA'' within 1 to 5 nucleotides,
preferably, 1 nucleotide, of the site to which SOL2"
hybridizes. The second oligonucleotide, OL2, is usually at
least as long as, and preferably longer than, SOL2'',
preferably, at least 2 nucleotides longer than SOL2''. In
general, the second oligonucleotide is about 20-100
nucleotides in length, preferably, 30-80 nucleotides in length
depending on the length of SOL2''. Normally, the second
oligonucleotide is chosen such that it dissociates from duplex
I'' at a higher temperature than that at which OL''
dissociates, usually at least 3°C, preferably, at least 5°C or
more higher.
The presence of OL2 in duplex I'' can effect the site of
cleavage of OL''. In particular, when OL2 binds to PA'' that
is not contiguous with the SOL2'' site of hybridization, the
cleavage site may be shifted one or more nucleotides.
The concentration of the second oligonucleotide employed
in this embodiment is usually at least 1 picomolar, but is
preferably above 0.1 nanomolar to facilitate rapid binding to
PA'', more preferably, at least 1 nanomolar to 1 micromolar.
In accordance with the embodiment of Fig. 3, OL'' in duplex
I'' is cleaved by 5'-endo to give DSOL2'' and LNSOL1''. The
reaction is permitted to continue until the desired number of
molecules of labeled fragment are formed. The reaction
parameters and detection of DSOL2'' and/or LNSOL1'' are
similar to those described above for the embodiment of Fig. 1.
In general and specifically in any of the embodiments of
Figs. 1 to 3 above, the 3'-end of the first oligonucleotide,
for example, OL, OL' and OL'', may have one or more
nucleotides that do not hybridize with the target
polynucleotide sequence and can serve as a label but need not
do so.
It is also within the purview of the present invention to
employ a single nucleoside triphosphate in any of the above
embodiments, depending on the particular
5'-endonuclease chosen for the above cleavage. The decision
to use a nucleoside triphosphate and the choice of the
nucleoside triphosphate are made empirically based on its
ability to accelerate the reaction in accordance with the
present invention. The nucleoside triphosphate is preferably
one that cannot be incorporated into the first oligonucleotide
as a consequence of the binding of the oligonucleotide to the
target polynucleotide sequence. In this particular embodiment
the added nucleoside triphosphate is present in a
concentration of 1 micromolar to 10 millimolar, preferably, 10
micromolar to 1 millimolar, more preferably, 100 micromolar to
1 millimolar. It is also within the purview of the present
invention to utilize the added nucleoside triphosphate to
chain extend the 3'-terminus of the second oligonucleotide to
render it contiguous with the site on the target
polynucleotide sequence at which the first oligonucleotide
hybridizes. In this approach the second oligonucleotide
serves as a polynucleotide primer for chain extension. In
addition, the nucleoside triphosphate is appropriately
selected to accomplish such chain extension and the
5'-nuclease is selected to also have template-dependent
nucleotide polymerase activity. In any event such an approach
is primarily applicable to the situation where the site of
binding of this second oligonucleotide, TPS2, is separated
from the site of binding of the first oligonucleotide, TPS1,
by a sequence of one or more identical bases that are
complementary to the added nucleotide triphosphate.
In the embodiment of Fig. 3 the mixture containing PA'',
OL'', the second oligonucleotide OL2 and the nucleoside
triphosphate is incubated at an appropriate isothermal
temperature at which OL'' and PA'' are in equilibrium with
duplex I'' wherein most of the molecules of PA'' and duplex
I'' are hybridized to OL2. During the time when a molecule
of OL'' is bound to PA'', the 5'-endo causes the cleavage by
hydrolysis of OL'' in accordance with the present invention.
When the remaining portion of cleaved oligonucleotide
(DSOL2'') dissociates from PA'', an intact molecule of OL''
becomes hybridized, whereupon the process is repeated.
In one experiment in accordance with the above
embodiment, incubation for 3 hours at 72°C resulted in the
production of over 1012 molecules of DSOL2'' and LNSOL1'',
which was over 104 increase over the number of molecules of
PA'' that was present initially in the reaction mixture. OL''
was labeled with a 32P-phosphate at the 5'-terminus. The
cleaved product LNSOL1'' was detected by applying the mixture
to an electrophoresis gel and detecting a band that migrated
more rapidly than the band associated with OL''. The
appearance of this band was shown to be associated with the
presence and amount of PA'' where a minimum of 108 molecules of
PA'' was detected.
Alternative approaches for detection of LNSOL1'' and/or
DSOL2'' may also be employed in the above embodiment. For
example, in one approach biotin is attached to any part of
SOL2'' that is cleaved from OL'' by the 5'-endonuclease. The
fragment DSOL2'' and OL'' containing the biotin are separated
from LNSOL1'', for example, by precipitation with streptavidin
and filtration. The unprecipitated labeled fragment LNSOL1''
is then detected by any standard binding assay, either without
separation (homogeneous) or with separation (heterogeneous) of
any of the assay components or products.
Homogeneous immunoassays are exemplified by enzyme
multiplied immunoassay techniques ("EMIT") disclosed in
Rubenstein, et al., U.S. Patent No. 3,817,837, column 3, line
6 to column 6, line 64; immunofluorescence methods such as
those disclosed in Ullman, et al., U.S. Patent No. 3,996,345,
column 17, line 59 to column 23; line 25; enzyme channeling
techniques such as those disclosed in Maggio, et al., U.S.
Patent No. 4,233,402, column 6, line 25 to column 9, line 63;
and other enzyme immunoassays such as the enzyme linked
immunosorbant assay ("ELISA") are discussed in Maggio, E.T.
supra. Exemplary of heterogeneous assays are the
radioimmunoassay, disclosed in Yalow, et al., J. Clin. Invest.
39:1157 (1960). The above disclosures are all incorporated
herein by reference. For a more detailed discussion of the
above immunoassay techniques, see "Enzyme-Immunoassay," by
Edward T. Maggio, CRC Press, Inc., Boca Raton, Florida, 1980.
See also, for example, U.S. Patent Nos. 3,690,834; 3,791,932;
3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654;
3,935,074; 3,984,533; 3,996,345; and 4,098,876, which listing
is not intended to be exhaustive.
Heterogeneous assays usually involve one or more
separation steps and can be competitive or non-competitive. A
variety of competitive and non-competitive assay formats are
disclosed in Davalian, et al., U.S.Patent No. 5,089,390,
column 14, line 25 to column 15, line 9, incorporated herein
by reference. A typical non-competitive assay is a sandwich
assay disclosed in David, et al., U.S. Patent No. 4,486,530,
column 8, line 6 to column 15, line 63, incorporated herein by
reference.
Another binding assay approach involves the luminescent
immunoassay described in U.S. Serial No. 07/704,569, filed May
22, 1991 entitled "Assay Method Utilizing Induced
Luminescence", which disclosure is incorporated herein by
reference.
As a matter of convenience, predetermined amounts of
reagents employed in the present invention can be provided in
a kit in packaged combination. A kit can comprise in packaged
combination (a) a first oligonucleotide having the
characteristic that, when reversibly hybridized to a portion
of a polynucleotide to be detected, it is degraded under
isothermal conditions by a 5'-nuclease to provide (i) a first
fragment that is substantially non-hybridizable to the
polynucleotide and (ii) a second fragment that is 3' of the
first fragment and is substantially hybridizable to the
polynucleotide, (b) a second oligonucleotide having the
characteristic of at least a portion thereof hybridizing to a
site on the polynucleotide that is 3' of the site at which the
first oligonucleotide hybridizes wherein the polynucleotide is
substantially fully hybridized to such portion of the second
oligonucleotide under isothermal conditions, and (c) the above
5'-nuclease. The kit can further comprise a single nucleoside
triphosphate.
The above kits can further include members of a signal
producing system and also various buffered media, some of
which may contain one or more of the above reagents. The
above kits can also include a written description of one or
more of the methods in accordance with the present invention
for detecting a polynucleotide analyte.
The relative amounts of the various reagents in the kits
can be varied widely to provide for concentrations of the
reagents which substantially optimize the reactions that need
to occur during the present method and to further
substantially optimize the sensitivity of any assay. Under
appropriate circumstances one or more of the reagents in the
kit can be provided as a dry powder, usually lyophilized,
including excipients, which on dissolution will provide for a
reagent solution having the appropriate concentrations for
performing a method or assay in accordance with the present
invention. Each reagent can be packaged in separate
containers or some reagents can be combined in one container
where cross-reactivity and shelf life permit.
EXAMPLES
The invention is demonstrated further by the following
illustrative examples. Temperatures are in degrees centigrade
(°C) and parts and percentages are by weight, unless otherwise
indicated.
EXAMPLE 1
A single stranded target DNA (2x108 molecules) (M13mp19
from Gibco, BRL, Bethesda, Maryland) (the "target DNA") was
combined with a 5'32P-labeled oligonucleotide probe, Probe 1,
(10uM) (5'CGT-GGG-AAC-AAA-CGG-CGG-AT3' (SEQ ID NO:1)
synthesized on a Pharmacia Gene Assembler (Pharmacia Biotech,
Piscataway, N.J.), an unlabeled oligonucleotide, Probe 2,
(1uM) (5'TTC-ATC-AAC-ATT-AAA-TGT-GAG-CGA-GTA-ACA-ACC-CGT-CGG-ATT-CTC3'
(SEQ ID NO:2) synthesized on a Pharmacia Gene
Assembler (Pharmacia Biotech), and 7.5 units of AmpliTaq DNA
polymerase (from Perkin-Elmer Corporation, Norwalk, N.J.) in
50uL of buffer (10mM Tris-HCl, pH 8.5, 50mM KCl, 7.5mM MgCl2,
100uM dATP). Probe 1 was a 20-base oligonucleotide that was
fully complementary to the target DNA and had a label on the
5'-nucleotide. Probe 2, the unlabeled probe, was designed to
anneal to the target DNA 3' to, and contiguous with, the site
at which the labeled probe annealed to the target DNA. The
dATP was shown to enhance the rate of cleavage by the
polymerase. However, good results were obtained in the
absence of dATP.
The reaction mixture was incubated at 72°C and
accumulation of product, a mononucleotide, namely,
5'32P-C-OH, was determined by visualization using
autoradiography following polyacrylamide gel electrophoresis.
The fold of amplification was determined by liquid
scintillation spectrometry of excised reaction products. A 105
fold amplification was observed.
The above reaction protocol was repeated using, in place
of Probe 1, a labeled probe, Probe 3, (5'TCG-TGG-GAA-CAA-ACG-GCG-GAT3'
(SEQ ID NO:3) prepared using a Pharmacia Gene
Assembler) that had 21 nucleotides with one base at the 5'-end
that was not complementary, and did not hybridize with, the
target DNA. The product of this reaction was a dinucleotide,
namely, 5'32P-TC-OH (SEQ ID NO:4), that represented a 105-fold
amplification.
The above reaction protocol was repeated with different
temperatures and different concentrations of reagents. All of
the reactions, including those mentioned above, were carried
out for a period of 3 hours. The following table summarizes
the reagents and reaction parameters and the results obtained
during the optimization procedure.
Probe | Probe (µM) | Target number | Taq (units) | Temp °C | Conditions | Fold amplification |
1 | 1 | 1010 | 2.5 | 72 | buffer as described; 1.5mM MgCl2 | 8.8 x 102 |
| 1 | 109 | | | | | | | 1.8 x 103 |
| 1 | 108 | ↓ | | | | | N.D. |
| 1 | 109 | 7.5 | | | ↓ | 2.0 x 103 |
| 1 | 109 | | | | | add dATP(100µM) | 1.4 x 103 |
| 1 | 108 | | | | | | | 1.0 x 104 |
| 10 | 109 | | | | | | | 1.4 x 104 |
| 10 | 108 | | | | | ↓ | 3.6 x 104 |
| 1 | 109 | | | | | increase MgCl2 (7.5mM) | 9.7 x 103 |
| 1 | 108 | | | | | | | 1.2 x 104 |
| 1 | 109 | | | | | | | 9.3 x 103 |
| 1 | 108 | | | | | | | 2.8 x 104 |
| 1 | 107 | | | ↓ | | | N.D. |
| 10 | 109 | | | 74 | | | 3.7 x 104 |
| 10 | 108 | | | | | | | 1.1 x 105 |
| 10 | 107 | | | ↓ | | N.D. |
3 | 1 | 109 | | | 72 | | | 9.9 x 103 |
| 1 | 108 | | | | | | | 2.6 x 104 |
| 1 | 107 | | | ↓ | | | N.D. |
| 10 | 109 | | | 74 | | | 4.6 X 104 |
| 10 | 108 | | | | | | | 1.0 X 105 |
| 10 | 107 | ↓ | ↓ | ↓ | N.D. |
EXAMPLE 2
The reaction protocol described in Example 1 was repeated
using the following probes in place of
Probe 1 or Probe 3:
Probe 4: 5'TTA-TTT-CGT-GGG-AAC-AAA-CGG-CGG-AT3' (SEQ ID
NO:5) (from Oligos Etc., Inc., Wilsonville, OR). Probe 4 had
26 nucleotides with six nucleotides at its 5'-end that were
not complementary, nor hybridizable with, the target DNA.
Probe 4 was present in a concentration of 1 micromolar. The
product of this reaction was an intact seven nucleotide
fragment, namely, 5'32P-TTATTTC-OH (SEQ ID NO:6), that
represented a 1.5X104-fold amplification. Probe 5: 5'GAT-TAG-GAT-TAG-GAT-TAG-TCG-TGG-GAA-CAA-ACG-GCG-GAT3'
(SEQ ID NO:7) was prepared using a Pharmacia Gene
assembler and had 39 nucleotides with 19 nucleotides at its
5'-end that were not complementary and did not hybridize with
the target DNA. The product of this reaction was an intact 20
nucleotide fragment, namely, 5'32P-GAT-TAG-GAT-TAG-GAT-TAG-TC-OH
(SEQ ID NO:8), that represented a 1.5X104-fold
amplification.
In repeating the above reactions in the absence of Probe
2, product was observed but the intensity of the spot on the
polyacrylamide gel was significantly less than in the presence
of Probe 2. Similar results were also observed where a 1
nucleotide space existed between the 3'-end of Probe 2 and the
second probe when both probes were hybridized to the target
DNA.
EXAMPLE 3
The reaction protocol described in Example 1 was repeated
using 2X10
9 target molecules and Probe 5 (see Example 2) at a
concentration of 1 micromolar in place of
Probe 1. The
reactions were conducted for three hours at a temperature of
72°C. using one of six different DNA polymerases, namely,
AmpliTaq DNA polymerase, Replitherm(TM) DNA polymerase
(Epicentre), Tfl(TM) DNA polymerase (Epicentre), Ultra
Therm(TM) DNA polymerase (Bio/Can Scientific), Thermalase
Tbr(TM) DNA polymerase (Amresco) and Panozyme(TM) DNA
polymerase. The product of the reaction was a 20-nucleotide
fragment (see Example 2). The following is a summary of the
results obtained.
Enzyme | Fragment (picomoles) |
AmpliTaq | 32 |
Replitherm | 18 |
Tfl | 5 |
Ultra Therm | 27 |
Tbr | 16 |
Panozyme | 25 |
The above experiments demonstrate that detectable
cleavage products were generated in a target-specific manner
at a single temperature using enzymes having 5'-nuclease
activity and a labeled oligonucleotide. The accumulation of
product was enhanced by the presence of a second
oligonucleotide that was longer than the first labeled
oligonucleotide and that was annealed to the target
polynucleotide sequence 3' of the site of hybridization of the
first labeled oligonucleotide. The reactions were carried out
at temperatures very close to the melting temperature (Tm) of
the labeled oligonucleotide with the target polynucleotide
sequence.
The above discussion includes certain theories as to
mechanisms involved in the present invention. These theories
should not be construed to limit the present invention in any
way, since it has been demonstrated that the present invention
achieves the results described.
The above description and examples fully disclose the
invention including preferred embodiments thereof.
Modifications of the methods described that are obvious to
those of ordinary skill in the art such as molecular biology
and related sciences are intended to be within the scope of
the following claims.